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1889-1895
0019-9567/94/$04.00+O
Copyright O 1994, American Society for Microbiology
Sample collection and nucleic acid extraction. The subgingival plaque sample (about 1 mg [wet weight]) was collected
with a calibrated curette from a deep periodontal pocket
(probing depth, >9 mm) of a 29-year-old white female patient
with severe destructive periodontitis. The material was gently
dispersed in 1 ml of prereduced broth and analyzed by
dark-field microscopy for the presence of motile spirochetes.
Approximately 20% of the bacteria were spirochetes. A 100-pl
aliquot of this suspension was frozen for subsequent in situ
hybridization analyses; the remaining part was centrifuged,
washed once in phosphate-buffered saline (PBS) (pH 7.0), and
*
Corresponding author. Present address: Universitatsklinikum
Charit6, Medizinische Fakultat der Humboldt-Universitat, Institut fur
Mikrobiologie und Hygiene. Clara-Zetkin-Str. 96, D-101 17 Berlin,
Germany. Phone: 49 030 220 24 11, ext. 280. Fax: 49 030 229 27 41.
1889
To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of a
patient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products of DNA and
RNA extracted from a subgingival plaque sample. Approximately 500 bp were amplified in PCRs by using
universally conserved primers with polylinker tails. Purified PCR products were cloned into Escherichia coli by
using the plasmid vector pUC19. The resultant clone library was screened by colony hybridization with a
radiolabeled, treponeme-specific oligonucleotide probe. The 16S rRNA inserts of 81 spirochetal clones were then
sequenced by standard procedures. Sequences were compared with 16S rRNA sequences of 35 spirochetes,
including the four known cultivable oral treponeme species. The analysis revealed an unexpected diversity of
oral treponemes from a single patient. When 98% or greater sequence similarity was used as the definition of
a species-level cluster, the clone sequences were found to represent 23 species. When 92% similarity was used
as the definition, the clones fell into eight major groups, only two of which contained named species, Treponema
vincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii were not represented
in any cluster. Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences. In situ
fluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of the
new species representing cluster 19 was used to identify cells of the target species directly in clinical samples.
1890
CHOI ET AL.
(5-bromo-4-chloro-3-indolyl-3-D-galactopyranoside)
(Boehringer), and 0.5 mM isopropylthiogalactoside (IPTG)
(Gibco BRL). Agar plates were incubated at 37C overnight.
Recombinants were selected on the basis of the white colony
phenotype.
Colony hybridization. White colonies were transferred with
sterile toothpicks to Luria-Bertani agar media supplemented
with ampicillin and then were lifted onto positively charged
nylon membranes (Biodyne B; Pall, Dreieich, Germany). Bacterial colonies were lysed according to standard procedures.
Oligodeoxynucleotide probes were 5' labeled with [T-32P]ATP
(5,000 Ci mmol'-; Amersham, Braunschweig, Germany) by
using T4 polynucleotide kinase (BRL) according to the supplier's recommendations. Free label was removed by passing
the reaction mixture over a Sephadex G-50 column (Pharmacia). Hybridization was performed essentially as described
previously (6).
Characterization of clones. The following probes (5, 8) were
used (hybridization temperatures are in parentheses): PI1V3
(Prevotella internedia specific), 5'-CAC GTG CCC CGC ITTT
ACT CCC CAA-3' (66C) (this sequence differs from a
sequence published by Dix et al. [5'-CAC GTG CCC CAC
rriT ACT CCC CAA-3'] (8) at one position [which is underlined]); P12V3 (Prevotella nigrescens specific), 5'-CGT GCG
CCA ATT TAT TCC CAC ATA-3' (60C); BFV3 (Bacteroides
forsythus specific), 5'-CGT ATC TCA TTT TAT TCC CCT
GTA-3' (56C); PGV324 (Porphyromonas gingivalis specific),
5'-CAA TAC TCG TAT CGC CCG TTA TTC-3' (60C); and
TREP (treponeme specific), 5'-GAC TTG CAT GCT TAA
(G/A)AC-3' (45C). One oligonucleotide complementary to
the successfully ligated polylinker-16S rRNA gene (rDNA)
junction was constructed to screen the clone library for true
recombinants; this was 5'-GGT CGA CAA CAG AGT TTG
AT-3' (48C) (the sequence complementary to the pUC19
polylinker is underlined). The TREP probe (base positions 46
to 63 according to E. coli numbering) includes three base
positions that are single-base signatures for spirochetes: U at
position 47, U at 50, and A at 52. The consensus base
signatures for other bacteria are C at 47, A at 50, and Y at 52
(45). While this probe has a single mismatch with some
spirochetes (A at position 53 versus G in leptospires), it has
four to five mismatches with essentially all other bacteria (31,
45).
Sequence and phylogenetic analysis. Plasmid minipreps
from TREP-positive recombinants were prepared as described
previously (38). Both insert strands were sequenced by a
modified Sanger dideoxy-chain-termination protocol using Sequenase (U.S. Biochemicals, Munich, Germany) according to
the manufacturer's suggestions. M13 reverse-sequencing
primer (5'-AAC AGC TAT GAC CAT G-3'; Pharmacia) and
X-Gal
INFEc-r. IMMUN.
Amplilication
method
PCR
RT-PCR9
NO. of
clones"
TREP"
577
5,841
PG"
Pid
PN"
BF/'
RESULTS
Identification of clones by hybridization with DNA probes.
A total of 6,418 randomly selected recombinants were transferred to nitrocellulose membranes for hybridization with a
panel of radiolabeled oligonucleotide probes. We chose this
rather large sample size to ensure that species representing less
than 0. I% of the population had a reasonable chance of being
represented by a clone. The numbers of clones hybridizing with
each probe are shown in Table 1.
Sequence and phylogenetic analysis of oral treponemes. A
total of 95 recombinants carrying treponeme-specific 16S
rRNA sequences were identified. Fourteen of these clones
were contaminated and were excluded from further study. The
remaining 81 clones were sequenced. Although complete 16S
sequences will be necessary for a thorough analysis of the
phylogeny of these new treponeme species, a comparison of
the 500-base partial sequences was sufficient to establish the
approximate phylogenetic positions of the clones as shown in
Fig. 1. Each of the 81 sequenced clones was presumptively
identified as a spirochete with the TREP probe. Spirochetal
identity was verified by phylogenetic analysis, thus validating
the specificity of the TREP probe. Seven of the 81 sequences
were chimeras and are not included in the figure. Chimeras
were found by noting that for some clones, the first half and the
second half of the sequence did not group together on the
phylogenetic tree. Examination of aligned sequences showed
that chimeras were composed of spirochetal and nonspirochetal halves. Thus, it may be useful in the future to use a
second probe to identify the 3' ends of amplified spirochetal
sequences. Lack of reactivity with this probe would indicate a
possible chimera. The sequences were also examined by using
the program CHECK-CHIMERA written by Niels Larsen
(29). This program identified as chimeric the same seven
sequences we had identified as chimeric.
The remaining 74 clones, derived by amplifying either DNA
or RNA, are shown as 53 nodes on the tree because a number
of nodes represent two or more identical sequences (the
number of identical sequences appears after the clone designation). 16S rRNA similarity for strains of a species is usually
greater than 99%. When a relaxed species threshold of approximately 98% similarity was used, the 74 clones were divided
into 23 clusters. When a threshold of approximately 92%
similarity was used, the 23 clusters fell into 8 major groups.
With the exception of cluster 23, which was related to the
leptospires, the clusters fell into the genus Treponema. T.
vincentii was represented by three clones in cluster 1, and T.
denticola was represented by two clones in cluster 11. There
were no clones related to T pectinovorum or T. socranskii.
Direct visualization of oral treponemes by in situ fluorescence hybridization. For future studies fluorescently labeled
oligonucleotides will be instrumental in assessing the role of
the newly identified spirochetes in the etiology of periodontal
infections. To demonstrate the feasibility of this approach, we
constructed a fluorescein isothiocyanate-labeled oligonucleotide probe specific for clone NZM399 (from cluster 19), which
was used to identify this novel treponeme in subgingival plaque
samples. As shown in Fig. 2, the fluorescein isothiocyanatelabeled probe ZM399 identified only one distinct spirochetal
morphotype, presumably the target species. No other bacteria
were stained.
DISCUSSION
Since most of the spirochetes in the oral cavity are presently
uncultivable, the nucleic acids of spirochetes from bacterial
plaque were analyzed directly by using molecular biological
techniques. The strategy was based upon studies of molecular
evolution using comparative sequence analysis of rRNAs (48).
Four different experimental approaches for obtaining rRNA
sequences have been described, which theoretically represent
without bias the entire population, including both cultivable
and uncultivable microorganisms. These approaches are as
follows: (i) direct sequencing of 5S rRNA (30), (ii) construction of cDNA libraries by using purified mixed-population
RNA as a template for primer-directed reverse transcription of
rRNAs (45), (iii) in vitro amplification of rRNA genes from
bulk DNA by using the PCR and rRNA-specific primers (13,
46), and (iv) construction of shotgun clone libraries from bulk
DNA and identification of rDNA-containing clones with a
mixed-kingdom probe (39). The latter approach is considered
to introduce the least bias into a phylogenetic analysis (39, 40).
Because of the minute amounts of DNA and RNA available
from subgingival plaque, we decided to establish a 16S rRNA
gene library by amplifying part of the 16S rRNA gene directly
from purified plaque DNA and from cDNA after reverse
transcription of plaque RNA. There were several reasons to
choose this dual approach. If DNA had been used exclusively,
there would have been a danger of amplifying silent rRNA
genes from bacteria carrying several rRNA operons. In addi-
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INFECT. IMMUN.
CHOI ET AL.
Cluster Group
92%
98X
(% Difference)
Xreeponeea vincentit
NZ.M3i00 R
NZM3!12 2P
,NZ3I 42 P
rNHZW397 R
_NZM360 R
NZM3105 A
NZM30464 2D0 R
NZM30292 0
NZM3103 R
NZM398 R
NZM3010 R
NZM3166
-13
4
-5
= 6
NZM334 R
NZM30378 0
NZM/30392 0
NZM3I4S R
NZM3f47 R
NZM3020 40
NZM30I
D
Treponema phagedenis
5R
NZM3106
NZM30472
NZM3042
II
NZM3!07 2R
NZM3i08 R
Jii
NZM3079
]2
III
13
NZM30527 0
NZM30298 2D. R
NZM302 17 0
NZM335 P
rreponema pall
Sp Jr oc hae ta steno s ,trepta
___I
ziaum
14
15
16
rhereophilic spirochete Hf
Spirochaeta zuelzerae
NZM304I0 3D
NZM3125 P
NZM3037 0
NZM(3I10 2R3R
NZM3122
NZM3I 13 2R
:7
-
NZM30505 0
1B
Treoonema brvantii
f
,,
NZM3i0i
NZM399
NZM3i57
0.
NZM3I24
NZM3ii8
NZM3i28
r NZM3109
NZM3155
2R
NZM3104
NZM30495
Treponema suc
rreponema socranski.i
Treponema pectino voruey
NZM30384
NZM30394
22
Treponema sp. CA
Irreponeaa saccharopbiulu
23
VII
VIII
ill lin.r
Leptospira biflexa
Leptospira Pnterrogans
FIG. 1. Phylogenetic tree of oral treponemes based on the comparison of partial 16S rRNA sequences. The scale bar represents a 10%
difference in nucleotide sequences as determined by measuring the lengths of the horizontal lines connecting two species. The letters following
clones indicate whether the sequence was obtained from DNA (D) or RNA (R). Numbers preceding the letters indicate the numbers of identical
clone sequences obtained.
R
NZM3081
NZM3.43 2R
NZM3i44
NZM3i38 R
NZM3158
1893
(29).
A fluorescently labeled, species-specific probe was used to
FIG. 2. In situ fluorescence hybridization with fluorescein isothiocyanate-labeled probe ZM399 for direct detection of the cluster 19 target
species in smears of formaldehyde-fixed subgingival plaque. Phase-contrast (A) and epifluorescence (B) microscopy results are shown.
1894
CHOI ET AL.
31:941-946.
12. Gersdorf, H., K. Pelz, and U. B. Gobel. 1993. Fluorescence in situ
hybridization for direct visualization of gram-negative anaerobes
in subgingival plaque samples. FEMS Immunol. Med. Microbiol.
6:109-114.
13. Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field.
1990. Genetic diversity in Sargasso Sea bacterioplankton. Nature
(London) 345:60-63.
14. Giovannoni, S. J., E. F. DeLong, G. J. Olsen, and N. R. Pace. 1988.
Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J. Bacteriol. 170:720-726.
15. Gobel, U. B., A. Geiser, and E. J. Stanbridge. 1987. Oligonucleotide probes complementary to variable regions of ribosomal RNA
discriminate between Mycoplasma species. J. Gen. Microbiol.
133:1969-1974.
16. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein
molecules, p. 21-132. In H. N. Munro (ed.), Mammalian protein
metabolism, vol. 3. Academic Press, Inc., New York.
REFERENCES
1. Amann, R., N. Springer, W. Ludwig, H. D. Gortz, and K. H.
Schleifer. 1991. Identification in situ and phylogeny of uncultured
bacterial endosymbionts. Nature (London) 351:161-164.
2. Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. Fluorescentoligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol.
172:762-770.
3. Armitage, G. C., W. R. Dickison, R. S. Jenderseck, S. M. Levine,
and D. W. Chambers. 1982. Relationship between the percentage
of subgingival spirochetes and severity of periodontal disease. J.
Periodontol. 53:550-556.
4. Chen, K., H. Neimark, P. Rumore, and C. R. Steinman. 1989.
Broad range DNA probes for detecting and amplifying eubacterial
nucleic acids. FEMS Microbiol. Lett. 57:19-24.
5. Choi, B.-K., and H. Gersdorf. Unpublished results.
6. Chuba, P. J., K. Pelz, G. Krekeler, T. S. De Isele, and U. Gobel.
1988. Synthetic oligodeoxynucleotide probes for the rapid detection of bacteria associated with human periodontitis. J. Gen.
Microbiol. 134:1931-1938.
7. DeLong, E. F., G. S. Wickham, and N. R. Pace. 1989. Phylogenetic
stains: ribosomal RNA-based probes for the identification of
single cells. Science 243:1360-1363.
8. Dix, K., S. M. Watanabe, S. McArdle, D. I. Lee, C. Randolph, B.
Moncla, and D. E. Schwartz. 1990. Species-specific oligodeoxynucleotide probes for the identification of periodontal bacteria.
J. Clin. Microbiol. 28:319-323.
9. Fraser, G. J., E. C. S. Chan, R. Siboo, F. E. Dewhirst, and B. J.
Paster. Phylogenetic position of new oral treponemes. J. Dent.
Res., in press.
10. Fuhrman, J. A., K. McCallum, and A. A. Davis. 1992. Novel major
archaebacterial group from marine plankton. Nature (London)
356:148-149.
11. Gersdorf, H., A. Meissner, K. Pelz, G. Krekeler, and U. B. Gobel.
1993. Identification of Bacteroides forsythus in subgingival plaque
from patients with advanced periodontitis. J. Clin. Microbiol.
INFEC-F. IMMUN.
40.
41.
42.
43.
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